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This article is from July/August 2008 and may contain outdated material.
Neurotrophic keratopathy is a degenerative disease of the corneal epithelium resulting from impaired corneal innervation. A reduction in corneal sensitivity or complete corneal anesthesia is the hallmark of this disease and is responsible for producing epithelial keratopathy, ulceration and perforation. Although numerous ocular and systemic diseases may result in neurotrophic keratopathy, there is one common insult: a lesion of the trigeminal nerve (cranial nerve V) or its branches.
Patients with neurotrophic keratopathy should undergo a complete medical and surgical history, a review of medications and an ocular examination. Although the clinical diagnosis may be made without difficulty, the management of neurotrophic keratopathy can be quite challenging.
Any condition affecting the trigeminal nerve or its branches can cause corneal anesthesia, resulting in neurotrophic keratopathy. The most common causes are herpes simplex and herpes zoster viral infections, followed by trigeminal neuralgia surgery and acoustic neuroma. During surgery, damage may occur to the trigeminal nucleus, root or ganglion, or to the ophthalmic branch of the nerve.
Toxicity from chronic use of topical ocular medications also may cause nerve damage and resultant corneal anesthesia. Indeed, topical anesthetics are a well-known cause of neurotrophic keratopathy. If a patient was recently diagnosed with a corneal abrasion and is a health care professional, the ophthalmologist must be suspicious for anesthetic abuse. Anesthetic abuse has also been linked to patients with psychiatric disease and/ or a history of drug abuse. Topical medications that may result in anesthesia include timolol, betaxolol, sulfacetamide and diclofenac sodium.
Another common etiology of neurotrophic keratopathy is diabetes mellitus. Diabetes either may be the primary cause of neurotrophic keratopathy or secondarily may predispose patients to this condition. Just as patients with long-standing diabetes may develop diabetic neuropathy in their hands and feet, a similar process can occur in the cornea, leading to sensory loss. Diabetic patients who undergo panretinal photocoagulation receive a secondary insult to the ciliary nerves. (For a complete list, see "Numerous Etiologies.")
The cornea has a high density of nerve endings from the long posterior ciliary nerves and is 100 times more sensitive than the conjunctiva. Studies have demonstrated that these sensory neurons directly influence the integrity of the corneal epithelium. In the presence of neuronal destruction, epithelial cells swell, lose microvilli and produce abnormal basal lamina. This can slow or halt mitosis, which leads to epithelial breakdown.
Additional studies have defined the role of neurotransmitters in the cornea, including acetylcholine, catecholamines, substance P, calcitonin gene-related peptide, neuropeptide Y, intestinal peptide, galanin and methionine-enkephalin. Mitosis of epithelial cells is increased by rising levels of intranuclear cyclic guanosine monophosphate (cGMP) and is decreased by rising levels of intracellular cyclic adenosine monophosphate (cAMP). Acetylcholine increases cGMP and therefore promotes epithelial growth. If this neurotransmitter is not released in the cornea, epithelial breakdown will result. Substance P also induces the proliferation of corneal epithelial cells.
A study in animals found that capsaicin (which depletes substance P) led to neurotrophic keratopathy, suggesting a trophic effect of substance P. In one clinical report, a patient with neurotrophic keratopathy demonstrated complete recovery after receiving a combination of substance P and insulin- like growth factor-1 eyedrops.1
Neurotrophic keratopathy can be divided into three stages based on the Mackie classification.
Stage 1 is characterized by mild, nonspecific signs and symptoms, including rose bengal staining of the inferior palpebral conjunctiva (the earliest sign). The viscosity of the tear mucus increases. There is decreased tear break-up time, leading to dry spots on the epithelium, which then stain with fluorescein, with resultant vascularization and scarring if the progression of neurotrophic keratopathy is not halted.2
Stage 2 involves a nonhealing corneal epithelial defect. The surrounding epithelium becomes loose, and Descemet's membrane develops folds as the stroma swells and becomes edematous. Characteristic of this stage, the defect forms a punched-out oval or circular shape. The edges of the defect may become smooth and rolled with time.
Stage 3 often ensues if stages 1 and 2 are not treated appropriately. It is characterized by stromal melting leading to perforation. The patient is often asymptomatic because of decreased corneal sensation.2
Examinations. Cranial nerve examination can help localize the cause of decreased corneal sensation. Dysfunction of cranial nerves VII and VIII may indicate an acoustic neuroma or damage from surgical resection of the lesion. Paresis of cranial nerves III, IV and VI may indicate an aneurysm or cavernous sinus pathology that also affects the trigeminal nerve. Adie's pupil has been associated with neurotrophic keratopathy.2
The eyelids should be examined carefully for both diagnostic and prognostic information. Eyelids that do not close properly may indicate a cranial nerve VII palsy. In addition, lagophthalmos leads to epithelial exposure and accelerates progression to stage 3 disease.
Corneal sensitivity is a vital piece of information and may be measured qualitatively with a piece of twisted cotton or quantitatively with a Cochet-Bonnet esthesiometer. This device quantifies corneal sensitivity by the length of a nylon filament required to initiate a blink or patient response. The nylon filament may be extended to as long as 6 cm. One study reported that only those patients with values of 2 cm or less developed epithelial sloughing and ulceration.1 It is important to remember that in some cases, such as herpes simplex and herpes zoster keratitis, the anesthesia of the cornea may be sectoral and therefore different quadrants of the cornea should be tested separately.
Slit-lamp and dilated funduscopic examinations must be performed in every patient and may give insight into the etiology. Corneal stromal scarring may indicate prior infection. Iris atrophy may be a sign of previous herpes infection. Dilated funduscopic examination may reveal diabetic retinopathy and/or extensive panretinal photocoagulation scars—known associations with neurotrophic keratopathy. There may be optic nerve pallor or swelling due to an intracranial tumor.
The diagnosis of neurotrophic keratopathy is generally made without difficulty based on history and examination findings. It is imperative to remember that the common finding in all cases is a decrease in corneal sensation. In stage 1 disease, punctate epithelial staining with fluorescein is a nonspecific sign and may be found in other conditions, including dry eye syndrome, blepharitis, chronic eye rubbing, exposure keratopathy, topical drug toxicity, ultraviolet keratopathy, mild chemical injury, contact lens– related disorders and corneal limbal stem cell deficiency. A careful medical and surgical history is important to sort through this differential diagnosis.
An ulcerated appearance of the cornea must raise suspicion for other causes, including infectious and immune etiologies. Appropriate cultures must be obtained and an immune work-up should be considered.
Once the diagnosis of neurotrophic keratopathy is established, treatment needs to be initiated immediately to prevent progression.
Stage 1 disease is generally treated with preservative-free artificial tears and ointments as well as consideration of punctal occlusion. Any current topical medications should be discontinued if possible.
Stage 2 epithelial defect must be treated in order to prevent a corneal ulcer from developing and to promote healing. Prophylactic antibiotic drops are generally added to the preservative-free artificial tears. A lateral tarsorrhaphy may be recommended, which may be effective in closing the epithelial defect. However, if the tarsorrhaphy is released too soon, epithelial breakdown will follow.
Other treatment options include an injection of botulinum A toxin into the upper eyelid levator muscle or amniotic membrane transplantation over the epithelial defect.
Stage 3 disease demands immediate attention in order to stop the stromal lysis and prevent perforation. In cases of stromal melting, topical collagenase inhibitors such as N-acetylcysteine, tetracycline or medroxyprogesterone may be administered.3
An article in the Japanese Journal of Ophthalmology reported on patients with persistent epithelial defects due to neurotrophic keratopathy. Patients were treated for 28 days with a substance P-derived peptide (FGLM)-amide and insulin-like growth factor (IGF-1). Complete epithelial resurfacing was achieved in eight of nine patients with no adverse effects.4
Corneas that appear to be very thin despite lubrication and tarsorrhaphy often require cyanoacrylate glue and a bandage contact lens. If perforation has already occurred, glue may be applied if the defect is less than 2 mm; otherwise a lamellar or penetrating keratoplasty is needed.2
Despite early and appropriate therapy, neurotrophic keratopathy may still progress to stage 3 disease. ___________________________
Dr. Wells is an ophthalmology resident and Dr. Michelson is a clinical associate professor of ophthalmology. Both are at the University of Alabama in Birmingham. ___________________________
1 Lambiase, A. et al. Curr Opinion Ophthalmol 1999;10:270–276. 2 Groos, E. Cornea 2004;94:1189–1196. 3 Bonini, S. et al. Eye 2003;17:989–995. 4 Nishida, T. et al. Jpn J Ophthalmol 2007;51(6):442–447.
INFECTIONHerpes simplexHerpes zosterLeprosy
TRIGEMINAL NERVE PALSYSurgery (as for trigeminal neuralgia)Neoplasia (such as acoustic neuroma) AneurysmsFacial trauma
CONGENITALFamilial dysautonomia (Riley-Day syndrome)Goldenhar-Gorlin syndromeMöbius' syndromeFamilial corneal hypesthesiaCongenital insensitivity to painwith anhidrosis
SYSTEMIC DISEASEDiabetes mellitusVitamin A deficiencyMultiple sclerosis
CORNEAL DYSTROPHIESLatticeGranular
IATROGENIC CAUSESContact lens wearTrauma to the ciliary nervesCorneal incisionsLASIK Panretinal laser photocoagulation
TOXIC CAUSESAnestheticsTimololBetaxololSulfacetamideDiclofenac sodiumChemical burnsCarbon disulfide exposureHydrogen sulfide exposure
MISCELLANEOUS CAUSESIncreasing ageAdie's pupilAny chronic condition causing corneal epithelial injury or inflammation


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Dance Theatre Of Harlem Company Returns
2012 marks the return of the groundbreaking Dance Theatre of Harlem Company with new dancers and a repertoire of exciting new commissions and DTH favorites. The company will reclaim its rightful place on the world's stages in the Fall of 2012, with works from the classical repertoire, Balanchine and world premiere contemporary ballets.
Starting January 14th, Dance Theatre of Harlem will host in-person auditions in San Francisco, Miami, Chicago and New York City. Online auditions are already open. For more information visit www.dancetheatreofharlem.org

subset: commoncrawl


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Please contact the Pellitteri the following day at 608-257-4285. We ask that you wait until the next day because collection may be delayed from your usual pick up time.
Show All Answers
Brush is picked up on the 2nd and 4th full week of each month. Please have your brush out at the curb by 7:00 am that Monday morning.
No, the city only picks up yard waste during our Fall Leaf Pickup. Please keep your yard waste separate from your brush pile and leaf pile during fall collection. You can always dispose of your yard waste at the city's Drop Off Site.


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One of the things I love about the blogging community is how there's such a diversity of approaches being tried by successful bloggers in their pursuits.
Take for instance two well known bloggers – Timothy Ferriss and Gary Vaynerchuk. Both take different approaches but both have been very successful in building strong online presence.
Timothy is famous for his book The 4-Hour Workweek a book looking at the simplification of life, outsourcing and focusing upon the important tasks at hand. Tim certainly works hard for his money but his approach is certainly a little different to Gary's.
For example Tim has written here at ProBlogger about how he finds that posting every 4-6 days on his blog is enough (and actually beneficial).
On the other hand Gary Vaynerchuck's inspirational keynote at Blog World Expo showed a different approach with a guy working massive hours, arguing that you should respond to every single email you get and that you need to be producing content every day.
Both of these guys have built successful businesses and great online presences through their blogging and social media (and I'm sure that there are some similarities between them also ) but both have done it differently.
To me this is encouraging. There are not 'formulas' and there is room for a diversity of approaches!
Which bloggers approach do you resonate with most – Gary or Tim?


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Daniel's Hill
Historical Summary
Developed as a residential area during the 1840s, Daniel's Hill was an elevated 900-1,000 acre tract of land overlooking the James River and Blackwater Creek. Originally, most of the land belonged to George Cabell, who built Point of Honor which today has been restored to its original form. The surrounding property passed out of Cabell's family and into the Daniel family through Judge William Daniel Jr., from whom the hill acquired its name. The main road leading up the spine of Daniel's Hill later came to be named after George Cabell.
Daniel's Hill exhibits a wide variety of architectural styles among its buildings constructed from the early to the late nineteenth century. The styles found among the larger homes include Federal, Italianate, Queen Anne, and Georgian Revival. The Daniel's Hill Historic District also contains unassuming mid-nineteenth to early-twentieth-century dwellings built as housing for workers. This hill was developed outside the Lynchburg city limits, but was annexed in 1870. Daniel's Hill is most prominently remembered for once being the epitome of Victorian elegance.
General Description
Daniel's Hill is a clearly defined linear district with Cabell Street, a heavily used traffic route, as the primary spine along the top of the ridge. Point of Honor, with major views over the City and the James River, denotes the southern end, while the northern boundary ends at "H" Street although the neighborhood continues beyond this point. Steep topography defines the east and west boundaries.
Daniel's Hill has a broad range of architecture including large mansions, modest dwellings, and vernacular cottages located in several sub-districts. The southern end and middle sections along Cabell Street contain high-style houses on mid to large size sites while the northern end contains more modest and smaller structures. A small neighborhood commercial area currently not being used as such is located at the corner of Cabell and "F" Streets. While generally built up, there are numerous underdeveloped or vacant sites reflecting numerous demolitions throughout the district. All of Daniel's Hill is currently zoned R-3.
Point of Honor
This important Federal residence is the focal point of the southern end of the district and has been restored as a city landmark. Dwellings on the back side of the lot were removed several years ago and the mansion is surrounded now by a block of open space with visitor parking along Norwood Street. Along the Cabell Street side is a row of frame vernacular houses of similar materials, size, scale, and setback.
Central Cabell Street
This sub-district extends from "B" Street to "D" Street along Cabell Street and includes larger-scale residences executed in a variety of architectural styles. There are several vacant lots in this district, as well as several large vacant structures poor in condition and in threat of demolition.
Cabell Street North
This sub-district includes several commercial structures at the "F' Street intersection of Cabell Street and continues to the end of the district at "H" Street. It contains numerous medium to smaller-scale vernacular structures and the altered Greek Revival residence, "Rivermont," one of the earliest structures of the district (circa 1857).
Stonewall Area
This small isolated sub-district consists of simple vernacular cottages on a steep hillside located below Cabell Street.
Norwood Street Area
This sub-district of small vernacular houses is part of a larger similar area along Norwood Street not currently a part of the historic district but which is a part of the neighborhood.
Description of Buildings
The 108 contributing structures that give character to Daniel's Hill vary tremendously in their style, scale, and sophistication. This district is a study in contrasts between the large-scale historic mansions of Cabell Street and the smaller worker residences scattered around the edges and on the side streets of the district. The dominant architectural style is the frame vernacular residence comprising 77% of all dwellings in the district. the remaining 23% reflect the variety of Cabell Street with several examples of Queen Anne, Italianate, Greek revival, and other nineteenth-century styles.
As in most of the historic districts in Lynchburg, there is almost equal division of structures with a horizontal (52%) or vertical (48%) emphasis on their facades.
Again, as in most districts, the overwhelming height of the buildings is two or more stories (88%) and the great majority of structures have front porches (79%).
In the building material category, 62% of the district buildings are framed, reflecting the large number of vernacular residences. The original fabric of 25% of the structures has been covered up with modern materials such as aluminum or imitation brick siding, the highest percentage of altered materials in all of the districts.
The majority of roofs in Daniel's Hill are standing seam metal (56%) with slate (25%) used primarily on the larger scaled structures. Roof forms are more varied with almost half (43%) using the gable design.
Setbacks are varied in the district with most of the smaller scaled structures tightly grouped and close to the street. Several of the Cabell Street mansions have much larger lots and generous setbacks from the thoroughfare.


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As we mark National Agriculture Day, I want to give special recognition to our farmers, ranchers and producers for their spirit of innovation. Too often, Americans don't take time to recognize the unique strength we have as a nation thanks to the innovation of American agriculture, and the willingness of our farmers, ranchers and producers to embrace new production methods.
We have a tremendously productive agriculture sector in the United States. In my lifetime, agriculture production has tripled. In 1950, a dairy cow produced about 5,300 pounds of milk each year; today, it's 22,000 pounds per year.
This innovation provides an abundant food supply, which keeps costs for all Americans low at the grocery store. In fact, we spend a lower percentage of our income on food than the people of any other developed nation. And thanks to the productivity of American agriculture, we're still able to export food around the world. The past four years have been the strongest in history for U.S. agricultural exports, which support more than a million jobs here at home.
At USDA, supporting innovation and productivity in agriculture is a mission-critical element of our work.
In recent years, USDA researchers and our university partners have helped uncover the genomes of pigs, turkeys, tomatoes, beans and more – all of which will help producers build on today's modern farming and ranching methods.
U.S. Forest Service researchers are looking into new uses for wood fibers in construction, body armor and other products, while investing in more than 150 groundbreaking projects to use wood as an energy source.
Our employees across the nation and around the world are working hard to ensure reliable delivery of the farm safety net, a robust Federal Crop Insurance program, open markets around the world and protection from invasive plants and animals.
And for the 98 percent of Americans who don't call the farm or ranch home, we've helped to strengthen understanding about agriculture. USDA's Know Your Farmer, Know Your Food effort has helped to strengthen local and regional food systems, and our Farm to School program is opening new doors to bring healthy, locally-sourced fruits and vegetables to our young people.
USDA is able to continue the most vital of these activities even as we implement across-the-board spending cuts, thanks to our efforts since 2009 to find more than $700 million in efficiencies. We've taken all of these steps because we deeply appreciate the work of America's farmers and ranchers – and we know that we have a critical duty to support their efforts.
There's no better time than today for all Americans to reflect on the contributions of American agriculture to the strength of our nation, and to say "Thank You" to farmers, ranchers and producers across the country. At USDA, we will keep doing our best to support their great work.
Stay tuned to the USDA blog this week for more Ag Day content from around USDA.


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Episode-44- Thoughts on the Differences between Permaculture and Agriculture
Podcast: Play in new window | Download (3.9MB)
In today's show we talk about land stewardship and permaculture as a true form of investing for the future. I also do a brief bit on politics today. A simple question, "how does Barak Obama define middle class income?", as in give us a number. Second a bit on on John McCain's pick of Alaska governor Sarah Palin as his V.P. running mate.
Please Note – I made a correction on today's show in audio and spliced it in but it will also be repeated here. For some reason I referred to Governor Palin as "Kathryn Palin" which of course is a mistake, her name is actually Sarah Palin. The fact that I podcast while in traffic during a commute sometimes may result in such errors, when I catch them I always make every attempt to correct them.
Tune in today and here my thoughts on…
Briefly on the Election
- What's up Obama, please give us an income figure for the term middle class
- Hey you too McCain, no politician should be able to say, "lower income", "middle class", etc with out numbers to go with them
- How the selection of Sarah Palin could almost guarantee a woman president in 2012
On the Main Subject
- The main difference between permaculture and agriculture
- Ideas for permaculture beyound tress and bushes
- Where wild game fits into your permaculture plan
- Handing down a family legacy with permaculture
- Growing grapes for sale to local wine makers
- Using permaculture crops to improve the quality of land
Resources for Today's Show


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Let $\mathcal F$ be a field. Suppose that there is a set $P \subset \mathcal F$ which satisfies the following properties:
For each $x \in \mathcal F$, exactly one of the following statements holds: $x \in P$, $-x \in P$, $x =0$.
For $x,y \in P$, $xy \in P$ and $x+y \in P$.
If such a $P$ exists, then $\mathcal F$ is an ordered field.
Define $x \le y \Leftrightarrow y -x \in P \vee x = y$.
Exercise: Prove that the field of complex numbers $\mathbb C$ cannot be given the structure of an ordered field.
My Work So Far: (Edit 1 note: This section and the Question is at the beginning, simply leaving this up for reference as to where I started)
Let $i$ be such that $i \in P, i \ne 0 \Rightarrow i > 0$. But $i^2 = -1 \notin P$.
My Question: I am not sure how much I need to redefine, and how I go about rigorously making this patchwork argument airtight. I am aware that I have not addressed how I assumed that $-1 \notin P$, but I'm not sure how to distinguish between $1$ and $i$ in this proof.
Edit #1
1st Step: Showing that $-1 \notin P$, observe that $(-1)(-1) = 1$ therefore if $-1 \in P$, both $x, -x \in P$, a contradiction.
2nd Step: To show $i \notin P$, we have that if $i \in P \Rightarrow i^2 \in P$, but $i^2 = -1 \notin P$, so $i \notin P$.
3rd Step: To show $-i \notin P$, we have $(-i)(-i) = i^2 \notin P$, so $-i$ cannot be in $P$.
Conclusion: Since $i \ne 0$, and $i, -i \notin P$, there is no set $P \subset \mathbb C$ that satisfies the above properties, thus $\mathbb C$ is not ordered.
Thank you André Nicolas and Eric Stucky for your help!


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Ivan Ljubicic gets past the second round of an ATP event for the first time since early February with his upset of 12th-seed Jo-Wilfried Tsonga in the second round of the Monte-Carlo Rolex Masters. Up next for the Croat is fifth seed Tomas Berdych; they're both in Nadal's quarter.
That Li Ning green makes him blend in with the background. Maybe ninja stealth is the way to success? FYI, Ljubicic is now ranked 40th in the world. Yikes.
(images via Getty Images)


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Antarctica is Earth's southernmost continent, overlying the South Pole. It is situated in the Antarctica region of the southern hemisphere, almost entirely south of the Antarctic Circle, and is surrounded by the Southern Ocean. At 14.4 million km2 (5.4 million sq mi), it is the fifth-largest continent in area after Asia, Africa, North America, and South America. About 98% of Antarctica is covered by ice, which averages at least 1.6 kilometers (1.0 mi) in thickness.
On average, Antarctica is the coldest, driest and windiest continent, and has the highest average elevation of all the continents. Antarctica is considered a desert, with annual precipitation of only 200 mm (8 inches) along the coast and far less inland. There are no permanent human residents, but anywhere from 1,000 to 5,000 people reside at the various research stations scattered across the continent throughout the year. Only cold-adapted plants and animals survive there, including penguins, seals, mosses, lichen, and many types of algae.
The name Antarctica is the romanized version of the Greek compound word ανταÏκτική (antarktiké), feminine of ανταÏκτικός (antarktikos), meaning "opposite to the north". Although myths and speculation about a Terra Australis ("Southern Land") date back to antiquity, the first confirmed sighting of the continent is commonly accepted to have occurred in 1820 by the Russian expedition of Mikhail Lazarev and Fabian Gottlieb von Bellingshausen.
The aurora australis, commonly known as the southern lights, is a glow observed in the night sky near the South Pole created by the plasma-full solar winds that pass by the Earth. Another unique spectacle is diamond dust, a ground-level cloud composed of tiny ice crystals. It generally forms under otherwise clear or nearly clear skies, so people sometimes also refer to it as clear-sky precipitation. A sun dog, a frequent atmospheric optical phenomenon, is a bright "spot" beside the true sun.
Visit wikipedia for more info.


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Copyright: © RWTHThe Faculty of Architecture today is located in the Reiff-Museum, which was named after Franz Reiff (1835-1902), Professor of Figure and Landscape Drawing and opened in 1908. On the one hand it served as a museum, on the other hand as a place where architects were trained. Its collection of almost 300 artworks included 200 original copies, which were used in the education of architects and art historians.
At RWTH Aachen University, architects have been trained since 1878, and urban planners since 1965, which placess it among the oldest university programs of this kind in Germany. Its long and – regarding the high number of prospective students – successful history originates from the profile of the education: At its center lies the conveyance of design, artistic and technical knowledge and skills based on social, cultural and environmental responsibility.
Due to the growing number of students and the resulting lack of space, the Reiff was extended by an annex during the years of 1960 to 1963. Further reconstructions and extensions followed shortly afterwards. After the death of Franz Reiff and his successor, Max Schmid-Burgks, the collection lost some of its repute, so that many works were given away, sold or forgotten. A project seminar at the Art History Institute was established in 2005, which refurbishes the collection. The collection of the Reiff Museum can be admired in a virtual museum. Reiff Museum.
With the Bologna reform, the Bachelor and Master system was also introduced in Aachen. Since the winter semester 2006/07, a Bachelor of Science in Architecture and since the winter semester 2009/10, a Master of Science in architecture and Urban planning have been offered. In the future, interdepartmental and interdisciplinary collaborations with many neighboring areas inside and outside of RWTH Aachen University.will help to further develop the university character of the education.


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For more coverage visit CNN affiliates WPVI and WABC. Are you there? Send pictures and video.
(CNN) -- Two packages found abroad that were bound for Jewish organizations in the United States contained a massive amount of explosive material that would have triggered a powerful blast, a source close to the investigation has told CNN.
U.S. officials believe that al Qaeda in the Arabian Peninsula, commonly referred to as AQAP, is behind the plot.
President Barack Obama confirmed that the packages -- intercepted in the United Kingdom and the United Arab Emirates -- originated in Yemen, the stronghold of al Qaeda in the Arabian Peninsula.
"We also know that al Qaeda in the Arabian Peninsula ... continues to plan attacks against our homeland, our citizens, and our friends and allies," he said during a press briefing on the incident.
In a statement Saturday, Dubai police said explosives in a package set to be shipped on a FedEx cargo plane had been "professionally" loaded in a package and connected via an electric circuit to a mobile phone chip hidden inside a printer.
"The plot style carries features similar to previous attacks carried out by terrorist organizations like Al Qaeda," the statement said according to WAM official news agency.
Another suspicious package, found at the UK's East Midlands Airport, contained a "manipulated" toner cartridge and had white powder on it as well as wires and a circuit board, a law enforcement source said.
The source close to the investigation said the type of material found in the devices was PETN, a highly explosive organic compound belonging to the same chemical family as nitroglycerin. Just six grams of PETN is enough to blow a hole in the fuselage of an aircraft.
PETN was allegedly one of the components of the bomb concealed by Umar Farouk AbdulMutallab, the Nigerian man accused of trying to set off an explosion aboard Northwest Airlines Flight 253 as it approached Detroit, Michigan, on December 25. AbdulMutallab is alleged to have been carrying 80 grams of PETN in that botched attack -- also believed to be the workings of al Qaeda in the Arabian Peninsula.
By comparison, the source said the two devices found Friday contained multiple times more PETN. The source also said it appears the devices were designed to be detonated by a cell phone with the help of a smaller amount of a second unidentified explosive substance.
The cell-phone theory was seconded by a wireless engineer for a major U.S.-based manufacturer, who analyzed a photo of one of the devices at CNN's request.
"This size and the shape of the PCB (printed circuit board) are typical to a handset cell phone type device," wrote Olivier Clerc, hardware application engineering manager for a cell-phone-parts manufacturer.
Both packages were bound for the United States, "specifically two places of Jewish worship in Chicago," Obama said.
The packages led to increased searches of cargo planes and trucks in several U.S. cities, said law enforcement sources with detailed knowledge of the investigation.
White House counterterrorism chief John Brennan said that "the materials that were found and the device that was discovered were intended to do harm."
Brennan said the discovery of the packages was made with help from Saudi Arabia, and issued a statement thanking the country for its "assistance in developing information that helped underscore the imminence of the threat emanating from Yemen."
A source with firsthand knowledge of the information told CNN that the Saudi Arabian government gave the United States tracking numbers of the two packages, allowing for quick tracing to the United Kingdom and Dubai.
Senate Intelligence Committee Chairwoman Dianne Feinstein, D-California, called the potential plot "a new novel thing -- and that is using FedEx and UPS planes to perhaps bring in something that might be explosive."
The Transportation Security Administration issued Friday afternoon a halt in the United States on all packages originating from Yemen, and shipping companies UPS, FedEx and DHL all said they were complying with the order.
Sen. Susan Collins, the ranking Republican on the Senate Homeland Security Committee, told CNN she is very concerned about holes in the system to screen cargo coming into the United States, said that had a credible intelligence source not warned about the suspicious packages, they may not have been detected with standard security procedures.
Collins, who was briefed by TSA chief John Pistole, said intelligence officials do not know yet if this was part of a larger plot, but she does believe al Qaeda is "continuing to test for vulnerabilities in our security system, and it appears we do have vulnerabilities in our system for transporting cargo."
After the packages were found Thursday night and Friday morning, authorities were tracking other packages shipped from Yemen in the same time frame, a law enforcement source said.
A Yemeni diplomat in Washington said that his government has opened a full-scale investigation into the incident but that it was too early to speculate or reach any conclusions.
Counterterrorism officials are taking the threat "very seriously," Obama said.
The Department of Homeland Security said it "had taken a number of steps to enhance security," including "heightened cargo screening and additional security at airports."
Some Jewish religious leaders in Chicago were alerted to the potential threat Friday, said Linda Haase, spokeswoman for the Jewish Federation of Metropolitan Chicago.
"We were notified about this earlier this morning," she said. "We are taking appropriate precautions, and we are advising local synagogues to do the same."
Rabbi Marvin Hier of the Simon Wiesenthal Center in Los Angeles, California, said that if synagogues were indeed the intended recipients of the packages, "this is just another indication of the dangerous world we live in where Jews are the principle target."
Meanwhile, U.S. authorities seemed most focused on inspecting cargo planes.
Investigators examined two UPS planes that landed at Philadelphia International Airport in Pennsylvania and another at Newark Liberty International Airport in New Jersey, said Mike Mangeot, a UPS spokesman. Authorities later gave the "all-clear" at both airports, the Transportation Security Administration said.
The TSA said authorities acted "out of an abundance of caution."
CNN's Paul Cruickshank, Eden Pontz, Mark Norman, Ross Levitt, Jeanne Meserve, Susan Candiotti, Caroline Paterson, Per Nyberg, Alan Duke, Steve Kastenbaum, Fran Townsend, Mike Ahlers, Carol Cratty, Chris Lawrence, Elise Labott, Pam Benson and Allan Chernoff contributed to this report.


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At the rate that Hollywood is plowing its way through Paul Verhoeven's sci-fi catalog, you'd expect Vegas bookies to start slashing the odds on an eventual "Starship Troopers" remake. Though it's only been two years since fanboys got their panties in a bunch over Len Wiseman's "Total Recall" reboot, many of those same fans have been dreading the release of the new "RoboCop." It will probably come as a surprise, then, that the film isn't nearly as bad as people feared it would be. In fact, it boasts a better cast, better effects and a better story, even if the 1987 original – which is admittedly pretty cheesy by today's standards – is still the better movie. So why bother with this remake? For starters, because it's not really a remake at all, instead taking the basic premise and carving its own path that falls more in line with current politics.
The year is 2028, and with the exception of the United States, the rest of the world is now policed by a robot military force operated by technology giant OmniCorp. The government has blocked the use of robots in the U.S. due to the belief that they can't be held accountable for killing, so OmniCorp CEO Raymond Sellars (Michael Keaton) decides to give the American public someone they can identify with by putting a man in a machine. And it's not long before they find the perfect subject when Detroit cop Alex Murphy (Joel Kinnaman) is critically injured in a car bombing after he's targeted by a local drug kingpin. With the help of Dr. Dennett Norton (Gary Oldman), a pioneer in robotic prosthetics, Sellars convinces Alex's wife, Clara (Abbie Cornish), that the procedure is the only way to keep him alive. But the very thing that makes Alex unique (his emotions) also affects his performance in the field, and when Norton tries to counteract that by programming his brain to act more like a machine, Alex's human side begins to fight back as he investigates his own murder.
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Posted in: Entertainment, Movie Reviews, Movies
Tags: Gary Oldman, Joel Kinnaman, Michael Keaton, RoboCop, RoboCop review


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- Research article
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Gene expression profiling of human mesenchymal stem cells derived from bone marrow during expansion and osteoblast differentiation
BMC Genomics volume 8, Article number: 70 (2007)
Human mesenchymal stem cells (MSC) with the capacity to differentiate into osteoblasts provide potential for the development of novel treatment strategies, such as improved healing of large bone defects. However, their low frequency in bone marrow necessitate ex vivo expansion for further clinical application. In this study we asked if MSC are developing in an aberrant or unwanted way during ex vivo long-term cultivation and if artificial cultivation conditions exert any influence on their stem cell maintenance. To address this question we first developed human oligonucleotide microarrays with 30.000 elements and then performed large-scale expression profiling of long-term expanded MSC and MSC during differentiation into osteoblasts.
The results showed that MSC did not alter their osteogenic differentiation capacity, surface marker profile, and the expression profiles of MSC during expansion. Microarray analysis of MSC during osteogenic differentiation identified three candidate genes for further examination and functional analysis: ID4, CRYAB, and SORT1. Additionally, we were able to reconstruct the three developmental phases during osteoblast differentiation: proliferation, matrix maturation, and mineralization, and illustrate the activation of the SMAD signaling pathways by TGF-β2 and BMPs.
With a variety of assays we could show that MSC represent a cell population which can be expanded for therapeutic applications.
In recent years mesenchymal stem cells (MSC) have generated a great deal of interest as a potential source for cell-based therapeutic strategies. Human MSC are easy to isolate from small aspirate of bone marrow via their adherence ability. These cells readily generate single-cell-derived colonies that can be highly expanded and differentiated into a variety of cell types, such as osteoblasts [1, 2], adipocytes [3], myocytes [4], astrocytes and neurons [5, 6]. Further, human MSC can improve cardiac function after infarction [7, 8] or symptoms of bone and cartilage defects [9–13], as well as neurodegenerative diseases such as Alzheimer's [14–16]. Their efficiency in multiple types of cellular therapeutic strategies has been demonstrated, including applications in treating children with osteogenesis imperfecta [17], hematopoietic recovery [18], and bone tissue regeneration [19, 20]. Also first preclinical trails are in progress to test their capacity and toxicity in applications for human treatment [21].
One great advantage of MSC is that these cells may be directly obtained from individual patients, thereby eliminating the complications associated with immune rejection of allogenic tissue and infectious diseases. However, for cell therapies MSC have to be expanded and/or manipulated to obtain a sufficient amount of cells that can be subsequently used for treatment. Despite growing experience and knowledge concerning human MSC and their use in cell-based strategies, the molecular mechanisms that govern MSC self-renewal, expansion and multilineage differentiation are not well understood and remain an active area of investigation.
In this study we asked if human MSC are developing in an aberrant or unwanted way during ex vivo long-term cultivation and if cultivation conditions exert any influence on their stem cell maintenance. To address this question systematically and comprehensively we first developed human oligonucleotide microarrays with 30.000 elements and then performed large-scale expression profiling of long-term expanded MSC isolated from clinically relevant samples. We monitored these cells during their expansion ex vivo with respect to proliferation kinetics, surface marker profile and differentiation potential. Finally we analyzed the gene expression profiles of MSC during osteogenic differentiation. Our results showed that expansion of MSC does not result in substantial genetic and morphological aberrations. We illustrated for the first time in a human model the three main stages of osteogenic development, and we could show the diverse regulation of the SMAD pathways by TGF-β2 and BMPs.
Human MSC maintain their undifferentiated phenotype during long-term expansion
The results of the ex vivo long-term expansion experiments showed that the undifferentiated phenotype of MSCs is maintained with respect to differentiation potential, surface marker profile and gene expression profiles. At confluence of 75–85% the cultivated cells were detached and flow cytometry analysis were performed to verify the purity of the cell population without any contaminations, such as haematopoietic cells. Following surface marker profile were detected: CD44+, CD90+, CD73+, CD105+, CD166+, CD11b-, CD34-, CD45-, CD117-, HLA DR-. For the subsequent experiments MSCs were expanded until the end of the tenth passage. At the end of each passage the cells were analyzed by flow cytometry and a cell aliquot was seeded out for testing their differentiation ability.
Proliferation kinetics and differentiation potential
The growth kinetics of five donors was investigated from the primary culture through the tenth passage, corresponding to approximately 26 cell doublings. For recalculating the starting number of MSC in the MNC fraction, CFU assays were performed and the MSC frequency was determined. Primary cultures reached their first confluence from around 80% in about 2 weeks and about 10 cell doublings. During the following passages the proliferation rate slowed down. During the whole expansion period – starting with passage two until passage ten – in every passage the osteogenic differentiation ability was tested. The osteogenic differentiation was confirmed by Alizarin Red S staining and Alkaline Phosphatase assay. Throughout this examination period differentiation ability into osteoblasts was observed in low passages (passage 2, after 12 cell doublings) as well as in a higher passage (passage 10, after 26 cell doublings), (Figure 1). MSC expanded to approximately 26 cell doublings were able to differentiate into adipocytes (Figure 1).
Surface marker profile
After the cultivation of MSC from the mononuclear cell fraction (passage 0) and at the end of each individual passage, the MSC were analyzed by flow cytometry to monitor the surface marker profile during their ex vivo expansion. Each investigated passage depicted homogenous cell population demonstrating that MSC did not alter their physical and morphological properties during high grade ex vivo expansion under the culture conditions tested [see Additional file 1]. In addition to the examination of the surface marker profile, we also performed Real-time RT-PCR Sybr™ Green I assays for stem cell specific genes. The examined stem cell markers, CD44, CD73, CD166, CD105, CD90 and STRO1 showed stable expression level during the whole expansion period proving the results obtained by FACS analyses. As expected, CD34, CD45 and TERT could not be detected on any examined time point [see Additional file 2].
Large-scale gene expression profiling of MSC during ex vivo expansion
In order to monitor the molecular events that govern the ex vivo expansion of undifferentiated MSC we performed large-scale gene expression profiling using oligonucleotide microarrays with 29952 elements (ArrayExpress, accession numbers A-MARS-2, E-MARS-5, and E-MARS-6, [see Additional file 3, 4, 5]). After background correction, global mean and dye swap normalization, and replicate handling, 838 genes could be detected as differentially expressed with at least 2-fold change, between cells from passage 2 with cells from passage 5, and 345 genes, when comparing passage 5 cells with passage 10 cells [see Additional file 6]. A modified version of the regularized t-test [22] with the complete data set was performed to identify significantly differentially expressed genes in ten examined donors (Nr. 1–10, passage 2 vs. passage 5) and five examined donors (Nr. 6–10, passage 5 vs. passage 10). The results showed that only 9 genes were significantly differentially expressed (p < 0.05) during 8 cell doublings when comparing cells from passage 2 versus cells from passage 5 (Table 1). Significantly differentially expressed genes represent proteins with diverse functions: extracellular matrix proteins (Col11A1, tenascin xb), proteins involved in bone, fat and epithelial cell metabolism (osteoprotegerin, b219ob receptor, FGF- 7), pain perception and stress response (proenkephalin), growth-promoting activity (IGF-2), and signaling and cell survival mechanisms (semaphorin 3c). The hypothetical protein XP_016240 is annotated as similar to keratinocyte growth factor-like protein, and the hypothetical protein dkfz434b044 is defined as a cysteine-rich secretory protein containing LCCL domain 2. The expression data from the above mentioned genes could be confirmed by independent real-time RT-PCR [see Additional file 7].
In sum, these results show that long-term expansion of MSC did not alter their differentiation potential, surface marker profiles, and expression profiles.
Expression profiling of MSC during osteogenic differentiation reveals distinct phases of osteogenic development
Osteogenic differentiation
Human MSC from three different donors (Nr. 9–11) were cultured until their fourth passage and were induced to osteogenic differentiation at a confluence of 70–80%. The osteogenic differentiation was observed during the whole period by microscopy and was terminated on day 21 after induction. First signs for calcification appeared as black regions within the cell monolayer after around seven days. The maximum of calcified extracellular matrix was observed after 21 days of treatment (Figure 1). The osteoblast phenotype was confirmed by Alkaline Phosphatase assay and Alizarin Red S staining (data not shown).
Expression of osteoblast specific marker genes
We performed Real-time RT-PCR assays to detect the expression levels of osteoblast specific marker genes MSX2 (homeobox gene MSX2), VDR (vitamin D receptor), COL1A1 (collagen 1A1), ALPL (alkaline phosphatase), SPARC (osteonectin), SPP1 (osteopontin) and BGLAP (osteocalcin) to prove the osteoblast phenotype. The log2 transformed results are shown in the supplementary material [see Additional file 8].
The data obtained by Real-time RT-PCR showed the expected expression profiles of the osteoblast phenotype. ALPL showed the expected expression level progression. Its expression was increased starting with day four of the differentiation period and decreased after day 14 until day 21 during the mineralization phase. The homeobox gene MSX2, which is implicated in osteoprogenitor cell function [23] and an up-stream regulator of RUNX2, which was described to be important for the osteogenic differentiation [24], were up-regulated during the whole period of differentiation. COL1A1 is known to be an early marker of osteoprogenitor cells [25]. Its maximum of expression was reached on day 21. Also SPARC showed a nearly constant, up-regulated expression level with its maximum on day 21 of differentiation. SPP1 is an extracellular matrix protein, known to peak twice in its expression [26]: around day 4, during proliferation, and between days 14–21, during mineralization. BGLAP was described as a late marker of developing osteoblasts appearing with matrix mineralization [27] and was maximally expressed on day 21. Interestingly, BGLAP was already significantly up-regulated on day four, whereas the mineralization could not be observed before day seven.
Large-scale gene expression profiling of MSC during osteogenesis
The RNA was harvested at four time points during the differentiation period and hybridized on human oligonucleotide microarrays. As reference, RNA harvested from MSC one day before the osteogenic induction was used (ArrayExpress, accession numbers A-MARS-2 and E-MARS-3). After LOWESS-subgrid normalization and filtering missing values 1108 genes were selected with two-fold up- or downregulated expression in at least one time point [see Additional file 9].
Correspondence between co-expressed genes and phenotypic changes
Differentially expressed genes were clustered according to their expression profiles using the k- means clustering method [28] [see Additional file 10]. The cluster analysis was performed with at maximum 50 iterations and using 12 different clusters, each containing between 58 and 167 genes (Figure 2). The number of clusters was estimated by FOM analyses [see Additional file 11]. Additionally, hierarchical clustering was performed and the results were comparable to k-means clustering [see Additional file 12].
By analyzing the genes of the different clusters and their expression profiles, we were able to reconstruct three main phases of osteogenic development (Figure 3): proliferation, matrix maturation, and mineralization. Additionally, novel candidate genes could be found which are not known to play a role during osteogenic differentiation: ID4 (inhibitor of DNA-binding), CRYAB (crystalline-αB) and SORT1 (sortillin1). The expression of these genes was subsequently confirmed by quantitative RT-PCR [see Additional file 13].
For gene ontology (GO) analysis we categorized ESTs with available RefSeq annotation (506 RefSeq annotations out of 1108 differentially expressed genes) according to GO terms for biological process (Figure 4), cellular component, and molecular function [see Additional file 14]. In detail, cluster 3 and 11 represented proliferation phase describing genes, which were involved in signal transduction, regulation of cell cycle, metabolism and regulation of transcription. Cluster 8 and 9 illustrated genes which were up-or down regulated during the whole period of differentiation. They describe the phase matrix maturation and are involved in development, cell differentiation, metabolism, regulation of DNA-dependent transcription. The phase of mineralization is evident in cluster 7 and 12, which include genes involved in signal transduction, transport, metabolism, and development.
The GO analysis showed that genes found in cluster 2 and 6 were also involved in cell adhesion, signal transduction, cell cycle, and regulation of transcription, similar to cluster 3 and 11. Cluster 10 described genes of development and metabolism among the rest. Cluster 1 and 5 combined genes of cell adhesion, transport, cell proliferation and regulation of transcription. Cluster 4 represented genes associated with transport and regulation of transcription.
In summary, GO annotation showed that nearly in each cluster genes of cell adhesion, signal transduction, transport, and regulation of transcription were present, illustrating the complex regulation of different events during osteogenesis.
Pathway analysis of the expression data set
The data set of 1108 genes with significant up or down regulation (+/- 2 fold, p ≤ 0.05) was mapped with the Pathway Explorer on pathways derived from the Biocarta and KEGG database [29, 30]. In total 276 out of 1108 genes could be mapped with their available RefSeq numbers (24.9%). Only pathways where at least 10% and more than five mapped genes could be found were selected for further examination. Following these criteria the TGF-β signaling pathway (12 mapped genes out of 116, 10.38%, see Figure 5) was ranked first, which summarized the different Smad pathways regulated by the members of the TGF-β family: BMP, TGF-β and inhibin.
Cell growth and proliferation is controlled by the members of the TGF-β superfamily which includes structurally related proteins like TGF-β, activins, and bone morphogenetic proteins (BMPs) [31–34]. In the here presented TGF-β signaling pathway BMP2 and BMP6, MADH1 and MADH9 (corresponding to SMAD1 and SMAD9) were up-regulated and the inhibitor of DNA binding ID4 was down-regulated (Figure 5). BMP2 and BMP6 are able to bind to BMPR-type I and II, whereas BMP6 is able to bind to ActRIIA [35, 36]. Due to the binding of these two molecules to the heterodimeric receptor complex the SMAD signaling pathway is activated. Our data illustrated further, that the inhibitor of DNA binding, ID4 was down regulated and the osteogenic differentiation could be induced. In parallel, TGF-β2, inhibin A and B were down-regulated during the differentiation period, whereby the following events were suppressed. Hence, under these experimental conditions the Smad-signaling pathway activated by BMP2 was responsible for the osteogenic development. In contrast, the activation of the Smad pathway by TGF-β and inhibin was suppressed.
In vivo relevance of human MSC as in vitro differentiation model
The in vitro differentiation of MSC into osteoblasts is often used model to examine the regulation mechanisms during osteoblast development. To evaluate the in vivo relevance of this model, we compared the RNA obtained at the diverse time points during osteogenic differentiation with RNA isolated from primary cultures of normal non-mineralized human osteoblasts derived from hipbone using our microarrays (ArrayExpress, accession numbers A-MARS-2, and E-MARS-4) [see Additional files 15, 16, 17]. The results showed that MSC on day seven of their differentiation were most similar to human osteoblasts [see Additional file 18]. This observation could be confirmed by the comparison of the morphology of the two cell types: Human osteoblasts isolated from hipbone represented non-mineralized osteoblasts with fibroblast-like phenotype, morphologically similar to induced MSC on day seven after induction.
In this study we could show for the first time that MSC maintain their undifferentiated phenotype after ex vivo long-term expansion. The results of the microarray assays showed that MSCs did not change significantly their gene expression profiles during long-term expansion since only nine out of almost 30.000 genes were differentially expressed between passage 2 and passage 5 in ten donors. In addition, no changes in the expression profiles after additional 5 passages could be observed in five of the ten donors. These surprising results can only be partially explained by the limitations of the microarray technology. Due to the lower sensitivity and uncomplete coverage of the transcriptome, some genes required for stem cell maintenance might have been missed. Additionally, individual differences between the donors and slightly different conditions during tissue extraction and sample treatment could increase the number identified genes which were otherwise filtered out during averaging. However, the microarray results were supported by the analyses of the effects of extensive long-term ex vivo cultivation of MSC on their proliferation capacity, their morphology, surface marker profile, and differentiation ability. During the long cultivation time we could observe, in concordance with other groups [37, 38], a progressive lost of proliferation ability and a slight shift in the cell-morphology of MSC of higher passage. However, their surface marker profile determined by flow cytometry (CD44+, CD90+, CD73+, CD105+, CD166+, CD45-, CD117-, HLA DR) and by Real-time RT-PCR was not altered. Over and above, the stem cell markers like CD90 and STRO1 showed stable expression levels, which further supports our observation that MSC maintained their undifferentiated phenotype and remained capable of osteogenic differentiation during ex vivo expansion at all tested passages (p2-p10).
Additionally to this clinically relevant evidence, we provide novel biological insights derived from the global view of the molecular processes during osteogenic development. First, we were able to illustrate the different phases of osteogenic development in a human model. Temporal expression of cell growth and osteoblast phenotype-related genes in a rodent model were first described by Stein et al. [39]. In our study, using large-scale expression profiling of clinical samples we could associate gene clusters to the three distinct phases of the development of osteoblast phenotype: proliferation, matrix maturation and mineralization.
Second, we could identify markers of late osteogenesis: ID4, CRYAB and SORT1:
a) ID4 is one of the four members of the ID (inhibitor of DNA binding) transcription factor family, which belongs to the basic-helix-loop-helix (bHLH) family. The ID members play a key role in differentiation processes and are involved in cell cycle control and cellular senescence [40–43]. It has been shown that ID proteins, by heterodimerization with bHLH proteins (MyoD, E12, E47), inhibit their binding to DNA, thus acting as dominant negative regulators, and have to be down-regulated to continue the differentiation process [44, 45]. To the best of our knowledge only ID1, ID2 and ID3 have been identified as most significantly up-regulated early targets of the osteogenic BMP2, BMP6 and BMP8 [46]. In our study we provide evidence that also ID4 exhibits a specific role during the osteogenic differentiation.
b) The second identified gene is crystallin-αB (CRYAB). Alpha crystallins are small heat shock proteins and composed of two gene products: alpha-A and alpha-B, for acidic and basic. Alpha-A is preferentially restricted to the vertebrate eye lens where it maintains the transparency and refractive index of the lens, whereas alpha-B is widely expressed in many tissues and organs [47]. The here observed up-regulation of CRYAB suggests an involvement in osteogenic differentiation but its specific role is still unknown. This involvement is supported by Furushima et al. [48], who performed a linkage study with knowledge-based candidate genes to detect genetic determinants associated with OPLL (ossification of the posterior longitudinal ligament of the spine), a predominant myelopathy among Japanese. The candidate genes for examination were obtained by cDNA microarray analysis of gene expression profiles during osteoblastic differentiation of MSC. Among the 24 genes identified in cDNA microarray analysis which could be associated with bone metabolism, CRYAB was the only gene which showed significant evidence of linkage.
c) Further, we could show the role of sortilin1 during osteogenic differentiation. Sortilin 1 (SORT1) represents a multi-ligand type-1 receptor which binds a number of unrelated ligands that participate in a wide range of cellular processes. The expression of SORT1 was increasing until day seven and then slowly decreasing. Sortilin1, also known as neurotensin receptor-3, is a glycoprotein originally purified from human brain [49]. The expression of sortilin1 is ubiquitous; it has been observed in heart, brain, placenta, lung, skeletal, muscle, pancreas, prostate, testis, small intestine, thyroid, and spinal cord, whereas its expression in bone marrow could not be detected [50]. The function of srotilin1 in bone metabolism is poorly understood. Maeda et al. [51] reported first differential expression during osteogenic differentiation and performed further experiments, which showed that sortilin 1 is promoting extracellular matrix maturation. Our results presented here strengthen this hypothesis, since sortilin 1 shows a similar expression progression as other well known osteogenic genes, such as ALPL or BMPs.
In summary, we have identified by expression profiling and confirmed by quantitative RT-PCR three markers of late osteogenesis. Further studies are necessary to quantify the expression of ID4, SORT-1 and CRYAB in primary human osteoblasts and other cell types and to identify the role of these genes in osteogenesis.
Third, by mapping the obtained expression data on curated biomolecular pathways, we were able to illustrate the distinct activation of the different Smad pathways induced by BMPs, TGF-β and inhibin, which were summarized as TGF-β pathways. The mapping of the expression data demonstrated that the fate of cell differentiation was simultaneously controlled at three different points: 1) BMP2, BMP6 and BMP8 activated the Smad-signaling pathway, consisting of the main components Smad 1, Smad 5 and Smad 8 resulting in osteoblast differentiation; 2) TGF-β2, regulating Smad 2 and Smad 4, was down regulated and the subsequent mechanisms were suppressed; and 3) inhibin was, like TGF-β2, down regulated. These findings have been confirmed by recent reports describing the diverse Smad-signaling pathways, but have never been illustrated in a general overview before this study [52–55].
Finally, the comparison of in vitro differentiated osteoblasts with in vivo developed osteoblasts on morphological and genetic level proved the usefulness of MSC as in vitro model for the investigation of the osteogenic development.
The here presented results demonstrate that MSC represent a cell population which can be expanded ex vivo and differentiated into osteoblasts. The genomic approach presented here represents a powerful tool to systematically and comprehensively investigate therapeutic approaches and hence, facilitate translational research on stem cells. However, it should be pointed out that new quality and safety standards have to be developed to guarantee risk free usage of ex vivo manipulated cell material for future therapeutic applications.
Cultivation and ex vivo expansion of human bone marrow-derived MSC and normal human osteoblasts
The mononuclear cell fractions (MNC) were derived from bone marrow from eleven different donors (Table 2), who gave consent after full information and approval by the hospital ethical committee (No. 12–091). All MNC were isolated from bone marrow aspirates at iliac crest during surgery. MNC were seeded at a density of 1 × 105 per cm2 and cultured for 24 hours. After these 24 hours non-adherent cells were removed by medium exchange. The adherent cells were cultured in expansion medium containing DMEM (Invitrogen), 10% FBS (selected lot, Stem Cell Technologies), 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine under a humidified atmosphere of 5% CO2 at 37°C. Medium was changed three times a week. After reaching a confluence of 75–85% the cells were detached with 0.05% trypsin/1 mM EDTA and replated at density of 4 × 103cells/cm2. The cells were expanded until the end of the tenth passage (corresponding to 26 cell doublings).
The cells for the cultivation of normal human osteoblasts derived from hipbone were obtained by PromoCell (Heidelberg, Germany). The cryopreserved normal human osteoblasts were thawed and cultivated until the end of the fourth passage following the protocols and media recommended by PromoCell.
Colony-forming-units of fibroblasts (CFU-F) assay
For determining the starting number of MSC in the mononuclear cell fraction CFU-F assays were performed. MNC from at least three donors were seeded in three concentrations, 2*106, 1*106 and 0.5*106 cells per T25 flasks, and incubated for 14 days in expansion medium without medium exchange under a humidified atmosphere of 5% CO2 at 37°C. After 14 days the cell suspension was removed, the adherent cells were fixed with methanol, dried and stained for 5 minutes with Giemsa staining solution (VWR). After washing with distilled water the colonies were counted and the human MSC frequency in the MNC fraction was calculated.
Ex vivo differentiation of human bone marrow-derived MSC in osteoblasts and adipocytes
For the induction of osteogenesis MSC were seeded at a density of 104 cells/cm2 in expansion medium. After 24 hours the differentiation was induced by medium exchange. The osteogenic differentiation medium was based on the expansion medium supplemented with 10 nM dexamethasone, 0.1 mM ascorbic-acid-2-phosphate and 10 mM β-glycerophosphate. The adipogenic differentiation medium contained in addition to the expansion medium 0.1 μM dexamethasone, 50 μM indomethacin and 5 μg/ml insulin. The media were changed three times a week. Osteogenic differentiation was detected by Alkaline Phosphatase assay (Sigma) and Alizarin Red S staining of mineralized matrix, whereas adipogenic differentiation was detected by Oil Red O staining of the adipocyte specific fat vacuoles.
The Alkaline Phosphatase assay was performed with the Alkaline Phosphatase kit No. 85 purchased by Sigma following the manufacturers' instructions. For the Alizarin Red S staining a 1% Alizarin Red S in 2% EtOH staining solution was prepared. The medium was removed; the cultures were washed twice with preheated 37°C PBS, fixed with 10% formaldehyde for 10 minutes, washed again with distilled water and incubated with the Alizarin Red S staining solution for 5 minutes. After incubation the staining solution was removed and the cultures were washed 5 times with distilled water to get rid of excessive color.
For the Oil Red O staining a 0.5% Oil Red O stock solution in 2-Propanol was prepared. For the staining procedure the stock solution was diluted 3:2 with distilled water, 10 minutes incubated at room temperature and filtrated by using Whatmanpaper #1. The medium was removed; the cells were washed with PBS, fixed with 10% formalin for at minimum 30 minutes, and stained with the Oil Red O staining solution for one hour.
Surface marker profiling by flow cytometry analysis
For flow cytometry analysis the cells were detached with 0.05% trypsin/EDTA and washed with PBS. 1 × 105 cells per tube were blocked for 5 minutes with human AB serum (Sigma) and stained for 15 minutes incubated in an ice bath in the dark with direct PE or FITC conjugated mouse anti-human monoclonal antibodies (Becton Dickinson) recognizing CD11b, CD34, CD38, CD44, CD45, CD73, CD90, CD117 and HLA-DR. After antibody incubation the cells were washed with PBS/sodium azide and resuspended in PBS in the needed volume for analyzing. 5 minutes before the analysis 7-AAD (0.25 μg per test) was applied to each sample for exclusion of dead cells. For negative control immunoglobulin isotype incubation was performed. FACS analyses were performed with FACS Calibur (Becton Dickinson).
Sample preparation – RNA isolation
Total RNA was isolated with TRIzol reagent (GibcoBRL-Life Technologies) following the instructions of the manufacturer. RNA concentration was determined by photometric measurement. The RNA quality was analyzed by Agilent 2100 Bioanalyzer RNA assays and evaluated by calculating the ratio of the 28S and 18S ribosomal RNA intensity peaks.
Microarray production
Human oligonucleotide microarrays were developed using 29550 oligonucleotides derived from 30 K MWG Human Oligo Set with 50 bp in length (provided by Prof. Reinhard Kofler, Tyrolian Cancer Research Institute), and dissolved in spotting buffer consisting of 3× SSC, 1.5 M Betaine. Microarrays were produced by spotting the reporters (including spotting buffer as negative control and human genomic DNA as positive control) in 48 blocks onto an epoxy-coated glass slide (Nexterion) resulting in 33456 features on a single high density array. Reporter molecules were fixed to the slides by baking at 42°C for 8 h at 50% relative humidity. RefSeq IDs indicate the genes represented by the oligonucleotides [see Additional file 3].
Microarray hybridization procedure
The used labeling and hybridization procedures were based on those developed at The Institute for Genomic Research (TIGR) and modified at the Institute for Genomics and Bioinformatics [see Additional file 4 and 5]. All hybridizations were repeated with reversed dye assignment (dye-swap). After hybridization slides were scanned with GenePix 4000B microarray scanner (Axon Instruments) at 10 μm resolution. Identical settings were used for scanning the corresponding dye-swapped hybridized slides. The resulting TIFF images for each of the two fluorophores were analyzed with GenePix Pro 4.1 (Axon Instruments).
Bioinformatics analyses
After image acquisition and filtering the data for low intensity, inhomogeneity and satured spots, the results files were normalized with the in-house developed software ArrayNorm [56]. After background correction the data sets were normalized by global-mean and dye-swap pairs or LOWESS-subgrid normalization. The obtained result files were used for further analyses such as cluster analysis [57], GO annotation [58] and pathway analyses [59]. Cluster analyses were performed with the Genesis software tool using k-means clustering function [28]. The number of clusters was varied from k = 1 to k = 15 and predictive power was analyzed with the figure of merit [60]. Subsequently, k = 12 was found optimal. GO annotation was performed on differentially expressed genes. Pathway analyses were performed with the in house developed Pathway Explorer software [59] which mapped the RefSeq IDs from the results file (cut-off level for differentially expressed genes: log2 ratio > 1) on public available pathways like Biocarta [61] and KEGG database [30]. All experimental parameters, images, raw and transformed data were uploaded to the microarray database MARS [62] and submitted via MAGE-ML export to a public repository (ArrayExpress, accession numbers A-MARS-2, E-MARS-3, E-MARS-4, E-MARS-5, and E-MARS-6) [63].
Real-time RT PCR
The same sources of total RNA used in the microarray experiments were used for the data validation by RT-PCR. The SuperScript™ II First Strand Synthesis System for RT PCR (Invitrogen) was used to synthesize cDNA of 5 μg RNA following manufacturers' recommendations. RNA contaminating genomic DNA was removed by treatment with DNAse I amplification grade (Invitrogen). The SuperScript™ II product was diluted 1:25 and directly used for the RT-PCR.
The RT-PCR assays were performed using the Eurogentec qPCR™ Mastermix Plus for Sybr™ Green I following manufacturer's recommendations. NO-reverse transcription Controls (No-RT) and No-Template Controls (NTC) were performed for each RNA type and primer pair. Cycling conditions on the ABI Prism 7000: 2 minutes at 50°C, 10 minutes at 95°C, 40 × (15 seconds at 95°C, 1 minute at 60°C) and the dissociation protocol at 60°C was added. For each sample RNA two cDNAs were prepared but in independent reverse transcription reactions. Each gene was tested with both cDNAs and the cDNAs were also tested in duplicate on one plate. Hence, four RT-PCR reactions were performed for the examination of one single gene. Further, the sample cDNA and the corresponding reference cDNA were tested on one plate, so that the data can be compared directly.
For normalization of the data the average of the expression level of four house-keeping genes, GAPDH, HPRT, beta-actin and beta-tubulin, was used.
Additional files
There are 18 additional files provided with the online version of this manuscript comprising raw data and results of the analyses. The data files are available on our web site http://genome.tugraz.at/MSC.
Haynesworth SE, Goshima J, Goldberg VM, Caplan AI: Characterization of cells with osteogenic potential from human marrow. Bone. 1992, 13: 81-88. 10.1016/8756-3282(92)90364-3.
Bruder SP, Jaiswal N, Haynesworth SE: Growth kinetics, self-renewal, and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem. 1997, 64: 278-294. 10.1002/(SICI)1097-4644(199702)64:2<278::AID-JCB11>3.0.CO;2-F.
Pittenger MF, Martin BJ: Mesenchymal stem cells and their potential as cardiac therapeutics. Circ Res. 2004, 95: 9-20. 10.1161/01.RES.0000135902.99383.6f.
Xu W, Zhang X, Qian H, Zhu W, Sun X, Hu J, Zhou H, Chen Y: Mesenchymal stem cells from adult human bone marrow differentiate into a cardiomyocyte phenotype in vitro. Exp Biol Med (Maywood). 2004, 229: 623-631.
Hofstetter CP, Schwarz EJ, Hess D, Widenfalk J, El M, Prockop DJ, Olson L: Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery. Proc Natl Acad Sci U S A. 2002, 99: 2199-2204. 10.1073/pnas.042678299.
Sanchez-Ramos J, Song S, Cardozo-Pelaez F, Hazzi C, Stedeford T, Willing A, Freeman TB, Saporta S, Janssen W, Patel N, Cooper DR, Sanberg PR: Adult bone marrow stromal cells differentiate into neural cells in vitro. Exp Neurol. 2000, 164: 247-256. 10.1006/exnr.2000.7389.
Hayflick L: Antecedents of cell aging research. Exp Gerontol. 1989, 24: 355-365. 10.1016/0531-5565(89)90043-0.
Shake JG, Gruber PJ, Baumgartner WA, Senechal G, Meyers J, Redmond JM, Pittenger MF, Martin BJ: Mesenchymal stem cell implantation in a swine myocardial infarct model: engraftment and functional effects. Ann Thorac Surg. 2002, 73: 1919-1925. 10.1016/S0003-4975(02)03517-8.
Bruder SP, Kraus KH, Goldberg VM, Kadiyala S: The effect of implants loaded with autologous mesenchymal stem cells on the healing of canine segmental bone defects. J Bone Joint Surg Am. 1998, 80: 985-996.
Kon E, Muraglia A, Corsi A, Bianco P, Marcacci M, Martin I, Boyde A, Ruspantini I, Chistolini P, Rocca M, Giardino R, Cancedda R, Quarto R: Autologous bone marrow stromal cells loaded onto porous hydroxyapatite ceramic accelerate bone repair in critical-size defects of sheep long bones. J Biomed Mater Res. 2000, 49: 328-337. 10.1002/(SICI)1097-4636(20000305)49:3<328::AID-JBM5>3.0.CO;2-Q.
Wakitani S, Goto T, Pineda SJ, Young RG, Mansour JM, Caplan AI, Goldberg VM: Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. J Bone Joint Surg Am. 1994, 76: 579-592.
Bruder SP, Jaiswal N, Ricalton NS, Mosca JD, Kraus KH, Kadiyala S: Mesenchymal stem cells in osteobiology and applied bone regeneration. Clin Orthop Relat Res. 1998, S247-S256. 10.1097/00003086-199810001-00025.
Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU: In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res. 1998, 238: 265-272. 10.1006/excr.1997.3858.
Woodbury D, Schwarz EJ, Prockop DJ, Black IB: Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res. 2000, 61: 364-370. 10.1002/1097-4547(20000815)61:4<364::AID-JNR2>3.0.CO;2-C.
Terada N, Hamazaki T, Oka M, Hoki M, Mastalerz DM, Nakano Y, Meyer EM, Morel L, Petersen BE, Scott EW: Bone marrow cells adopt the phenotype of other cells by spontaneous cell fusion. Nature. 2002, 416: 542-545. 10.1038/nature730.
Dezawa M, Takahashi I, Esaki M, Takano M, Sawada H: Sciatic nerve regeneration in rats induced by transplantation of in vitro differentiated bone-marrow stromal cells. Eur J Neurosci. 2001, 14: 1771-1776. 10.1046/j.0953-816x.2001.01814.x.
Horwitz EM, Gordon PL, Koo WK, Marx JC, Neel MD, McNall RY, Muul L, Hofmann T: Isolated allogeneic bone marrow-derived mesenchymal cells engraft and stimulate growth in children with osteogenesis imperfecta: Implications for cell therapy of bone. Proc Natl Acad Sci U S A. 2002, 99: 8932-8937. 10.1073/pnas.132252399.
Koc ON, Gerson SL, Cooper BW, Dyhouse SM, Haynesworth SE, Caplan AI, Lazarus HM: Rapid hematopoietic recovery after coinfusion of autologous-blood stem cells and culture-expanded marrow mesenchymal stem cells in advanced breast cancer patients receiving high-dose chemotherapy. J Clin Oncol. 2000, 18: 307-316.
Quarto R, Mastrogiacomo M, Cancedda R, Kutepov SM, Mukhachev V, Lavroukov A, Kon E, Marcacci M: Repair of large bone defects with the use of autologous bone marrow stromal cells. N Engl J Med. 2001, 344: 385-386. 10.1056/NEJM200102013440516.
Petite H, Viateau V, Bensaid W, Meunier A, de Pollak C, Bourguignon M, Oudina K, Sedel L, Guillemin G: Tissue-engineered bone regeneration. Nat Biotechnol. 2000, 18: 959-963. 10.1038/79449.
Lazarus HM, Haynesworth SE, Gerson SL, Rosenthal NS, Caplan AI: Ex vivo expansion and subsequent infusion of human bone marrow-derived stromal progenitor cells (mesenchymal progenitor cells): implications for therapeutic use. Bone Marrow Transplant. 1995, 16: 557-564.
Baldi P, Long AD: A Bayesian framework for the analysis of microarray expression data: regularized t -test and statistical inferences of gene changes. Bioinformatics. 2001, 17: 509-519. 10.1093/bioinformatics/17.6.509.
Cheng SL, Shao JS, Charlton-Kachigian N, Loewy AP, Towler DA: MSX2 promotes osteogenesis and suppresses adipogenic differentiation of multipotent mesenchymal progenitors. J Biol Chem. 2003, 278: 45969-45977. 10.1074/jbc.M306972200.
Satokata I, Ma L, Ohshima H, Bei M, Woo I, Nishizawa K, Maeda T, Takano Y, Uchiyama M, Heaney S, Peters H, Tang ZQ, Maxson R, Maas R: Msx2 deficiency in mice causes pleiotropic defects in bone growth and ectodermal organ formation. Nature Genetics. 2000, 24: 391-395. 10.1038/74231.
Jikko A, Harris SE, Chen D, Mendrick DL, Damsky CH: Collagen integrin receptors regulate early osteoblast differentiation induced by BMP-2. Journal of Bone and Mineral Research. 1999, 14: 1075-1083. 10.1359/jbmr.19188.8.131.525.
J.E. A: Regulation of osteoblast formation and function. Rev Endocr Metab Disord. 2001, 2: 81-94. 10.1023/A:1010011209064.
Aubin JE: Bone stem cells. Journal of Cellular Biochemistry. 1998, 73-82. 10.1002/(SICI)1097-4644(1998)72:30/31+<73::AID-JCB11>3.0.CO;2-L.
Hartigan JA: Clustering. Annu Rev Biophys Bioeng. 1973, 2: 81-101. 10.1146/annurev.bb.02.060173.000501.
Biocarta pathway database. 2005, [http://www.biocarta.com/genes/index.asp]
Kanehisa M, Goto S, Kawashima S, Nakaya A: The KEGG databases at GenomeNet. Nucleic Acids Res. 2002, 30: 42-46. 10.1093/nar/30.1.42.
Massague J: TGF-beta signal transduction. Annu Rev Biochem. 1998, 67: 753-791. 10.1146/annurev.biochem.67.1.753.
Zwijsen A, Verschueren K, Huylebroeck D: New intracellular components of bone morphogenetic protein/Smad signaling cascades. FEBS Lett. 2003, 546: 133-139. 10.1016/S0014-5793(03)00566-0.
Wan M, Cao X: BMP signaling in skeletal development. Biochem Biophys Res Commun. 2005, 328: 651-657. 10.1016/j.bbrc.2004.11.067.
Ducy P, Karsenty G: The family of bone morphogenetic proteins. Kidney Int. 2000, 57: 2207-2214. 10.1046/j.1523-1755.2000.00081.x.
Banfi A, Muraglia A, Dozin B, Mastrogiacomo M, Cancedda R, Quarto R: Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: Implications for their use in cell therapy. Exp Hematol. 2000, 28: 707-715. 10.1016/S0301-472X(00)00160-0.
Bruder SP, Kurth AA, Shea M, Hayes WC, Jaiswal N, Kadiyala S: Bone regeneration by implantation of purified, culture-expanded human mesenchymal stem cells. J Orthop Res. 1998, 16: 155-162. 10.1002/jor.1100160202.
Stein GS, Lian JB: Molecular mechanisms mediating proliferation/differentiation interrelationships during progressive development of the osteoblast phenotype. Endocr Rev. 1993, 14: 424-442. 10.1210/er.14.4.424.
Kreider BL, Benezra R, Rovera G, Kadesch T: Inhibition of myeloid differentiation by the helix-loop-helix protein Id. Science. 1992, 255: 1700-1702. 10.1126/science.1372755.
Zebedee Z, Hara E: Id proteins in cell cycle control and cellular senescence. Oncogene. 2001, 20: 8317-8325. 10.1038/sj.onc.1205092.
Ruzinova MB, Benezra R: Id proteins in development, cell cycle and cancer. Trends Cell Biol. 2003, 13: 410-418. 10.1016/S0962-8924(03)00147-8.
Norton JD: ID helix-loop-helix proteins in cell growth, differentiation and tumorigenesis. J Cell Sci. 2000, 113 ( Pt 22): 3897-3905.
Benezra R, Davis RL, Lockshon D, Turner DL, Weintraub H: The protein Id: a negative regulator of helix-loop-helix DNA binding proteins. Cell. 1990, 61: 49-59. 10.1016/0092-8674(90)90214-Y.
Pagliuca A, Cannada-Bartoli P, Lania L: A role for Sp and helix-loop-helix transcription factors in the regulation of the human Id4 gene promoter activity. J Biol Chem. 1998, 273: 7668-7674. 10.1074/jbc.273.13.7668.
Peng Y, Kang Q, Luo Q, Jiang W, Si W, Liu BA, Luu HH, Park JK, Li X, Luo J, Montag AG, Haydon RC, He TC: Inhibitor of DNA binding/differentiation helix-loop-helix proteins mediate bone morphogenetic protein-induced osteoblast differentiation of mesenchymal stem cells. J Biol Chem. 2004, 279: 32941-32949. 10.1074/jbc.M403344200.
Derham BK, Harding JJ: Alpha-crystallin as a molecular chaperone. Prog Retin Eye Res. 1999, 18: 463-509. 10.1016/S1350-9462(98)00030-5.
Furushima K, Shimo-Onoda K, Maeda S, Nobukuni T, Ikari K, Koga H, Komiya S, Nakajima T, Harata S, Inoue I: Large-scale screening for candidate genes of ossification of the posterior longitudinal ligament of the spine. J Bone Miner Res. 2002, 17: 128-137. 10.1359/jbmr.2002.17.1.128.
Petersen CM, Nielsen MS, Nykjaer A, Jacobsen L, Tommerup N, Rasmussen HH, Roigaard H, Gliemann J, Madsen P, Moestrup SK: Molecular identification of a novel candidate sorting receptor purified from human brain by receptor-associated protein affinity chromatography. J Biol Chem. 1997, 272: 3599-3605. 10.1074/jbc.272.6.3599.
Maeda S, Nobukuni T, Shimo-Onoda K, Hayashi K, Yone K, Komiya S, Inoue I: Sortilin is upregulated during osteoblastic differentiation of mesenchymal stem cells and promotes extracellular matrix mineralization. J Cell Physiol. 2002, 193: 73-79. 10.1002/jcp.10151.
Nohe A, Hassel S, Ehrlich M, Neubauer F, Sebald W, Henis YI, Knaus P: The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways. J Biol Chem. 2002, 277: 5330-5338. 10.1074/jbc.M102750200.
Afzal F, Pratap J, Ito K, Ito Y, Stein JL, van Wijnen AJ, Stein GS, Lian JB, Javed A: Smad function and intranuclear targeting share a Runx2 motif required for osteogenic lineage induction and BMP2 responsive transcription. J Cell Physiol. 2005, 204: 63-72. 10.1002/jcp.20258.
ten Dijke P, Goumans MJ, Itoh F, Itoh S: Regulation of cell proliferation by Smad proteins. J Cell Physiol. 2002, 191: 1-16. 10.1002/jcp.10066.
Pieler R, Sanchez-Cabo F, Hackl H, Thallinger GG, Trajanoski Z: ArrayNorm: comprehensive normalization and analysis of microarray data. Bioinformatics. 2004, 20: 1971-1973. 10.1093/bioinformatics/bth174.
Sturn A, Quackenbush J, Trajanoski Z: Genesis: cluster analysis of microarray data. Bioinformatics. 2002, 18: 207-208. 10.1093/bioinformatics/18.1.207.
Harris MA, Clark J, Ireland A, Lomax J, Ashburner M, Foulger R, Eilbeck K, Lewis S, Marshall B, Mungall C, Richter J, Rubin GM, Blake JA, Bult C, Dolan M, Drabkin H, Eppig JT, Hill DP, Ni L, Ringwald M, Balakrishnan R, Cherry JM, Christie KR, Costanzo MC, Dwight SS, Engel S, Fisk DG, Hirschman JE, Hong EL, Nash RS, Sethuraman A, Theesfeld CL, Botstein D, Dolinski K, Feierbach B, Berardini T, Mundodi S, Rhee SY, Apweiler R, Barrell D, Camon E, Dimmer E, Lee V, Chisholm R, Gaudet P, Kibbe W, Kishore R, Schwarz EM, Sternberg P, Gwinn M, Hannick L, Wortman J, Berriman M, Wood V, de la CN, Tonellato P, Jaiswal P, Seigfried T, White R: The Gene Ontology (GO) database and informatics resource. Nucleic Acids Res. 2004, 32: D258-D261. 10.1093/nar/gkh066.
Mlecnik B, Scheideler M, Hackl H, Hartler J, Sanchez-Cabo F, Trajanoski Z: PathwayExplorer: web service for visualizing high-throughput expression data on biological pathways. Nucleic Acids Res. 2005, 33: W633-W637. 10.1093/nar/gki391.
Yeung KY, Haynor DR, Ruzzo WL: Validating clustering for gene expression data. Bioinformatics. 2001, 17: 309-318. 10.1093/bioinformatics/17.4.309.
Maurer M, Molidor R, Sturn A, Hartler J, Hackl H, Stocker G, Prokesch A, Scheideler M, Trajanoski Z: MARS: microarray analysis, retrieval, and storage system. BMC Bioinformatics. 2005, 6: 101-101. 10.1186/1471-2105-6-101.
Brazma A, Parkinson H, Sarkans U, Shojatalab M, Vilo J, Abeygunawardena N, Holloway E, Kapushesky M, Kemmeren P, Lara GG, Oezcimen A, Rocca-Serra P, Sansone SA: ArrayExpress--a public repository for microarray gene expression data at the EBI. Nucleic Acids Res. 2003, 31: 68-71. 10.1093/nar/gkg091.
We thank Hubert Hackl for assistance with the additional data files, Bernhard Mlecnik for the pathway analyses, and Robert Molidor for the microarray database support. This work was supported by the Christian-Doppler Laboratory for Genomics and Bioinformatics, and the GEN-AU projects Bioinformatics Integration Network (BIN) and Genomics of Lipid-Associated Disorders (GOLD).
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BK performed the experiments and analyzed the data. FSC was responsible for the microarray data normalization and statistical analysis. GF isolated the bone marrow aspirates. RW was responsible for the clinical studies. AJ and KHP supported the design of the experiments and the interpretation of the results. MS, CP, and AP developed and produced the microarrays. ZT was responsible for the overall conception and project coordination. All authors gave final approval of the version to be published.
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Additional File 2: Expression of stem cell specific marker genes evaluated by Realtime RT-PCR. Expression of stem cell specific marker genes evaluated by Realtime RT-PCR. (PDF 9 KB)
Additional File 4: SOP AMINOALLYL LABELING OF RNA FOR HUMAN OLIGO CHIPS. Standard Operating Procedure for AMINOALLYL LABELING OF RNA FOR HUMAN OLIGO CHIPS. (PDF 36 KB)
Additional File 5: SOP HUMAN OLIGO CHIP PROBE HYBRIDIZATION. Standard Operating Procedure for microarray hybridization. (PDF 87 KB)
Additional File 6: Differentially expressed ESTs. Differentially expressed ESTs (>2-foldchange) when comparing p2/p5 and p5/p10 for following moderated t-test (XLS 302 KB)
Additional File 7: Validation of microarray data by Realtime RT-PCR. Gene expression analysis of long-term cultivated MSC. (PDF 14 KB)
Additional File 8: Gene expression of osteoblast specific genes determined by Real-time RT-PCR. Gene expression of osteoblast specific genes determined by Real-time RT-PCR to prove the osteogenic phenotype. (PDF 8 KB)
Additional File 13: Validation of microarray data using real-time RT-PCR. Gene expression analysis of MSC during osteogenesis. (PDF 63 KB)
Additional File 18: Evaluation of human MSC as in vitro differentiation model. Comparison of human osteoblasts with in vitro differentiated hMSC. (PDF 8 KB)
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Kulterer, B., Friedl, G., Jandrositz, A. et al. Gene expression profiling of human mesenchymal stem cells derived from bone marrow during expansion and osteoblast differentiation. BMC Genomics 8, 70 (2007). https://doi.org/10.1186/1471-2164-8-70
DOI: https://doi.org/10.1186/1471-2164-8-70